The ultimate goal of crossing over during prophase I is to tether homologous chromosomes together until the first meiotic division, when they must then segregate equally into daughter cells that then enter meiosis II. The importance of this stage is highlighted by the fact that approximately 50% of all spontaneous miscarriages are due to non-disjunction errors at the first meiotic division, while 90% of Down syndrome cases can be attributed to errors in maternal meiosis. The MutL homologs of the DNA mismatch repair (MMR) family, MLH1 and MLH3, localize along meiotic chromosomes during prophase I in yeast, zebrafish, mouse and human. In the mouse, these proteins localize together at the late meiotic nodules that will eventually go on to become mature crossovers, and they serve to ensure that these chiasmata remain intact until entry in to diplonema. Thus both proteins are essential for normal meiotic progression as demonstrated by the sterility of mice bearing mutations in Mlh1 or in Mlh3. The aim of the current proposal is to understand how the MLH1-MLH3 heterodimer regulates the placement and integrity of crossovers in mammalian germ cells with the hypothesis that unique regulatory factors, both intrinsic and extrinsic to MLH1-MLH3, are in place to ensure the appropriate levels of crossing over. Aim 1 is focused on exploring the "intrinsic" regulation of MLH1-MLH3 function, by analysis of Mlh1-ATPase deleted mice, and by the generation and analysis of Mlh3-Endonuclease domain deleted mice. In Aim 2, we will explore the extrinsic regulation of MLH1-MLH3 by focusing on their associations with two major components of the recombinogenic machinery, Bloom syndrome mutated (BLM) protein and MUS81, both of which are known to interact functionally with each other and with the MMR pathway in many systems. Interactions between MLH1-MLH3 and BLM will be tested by analyzing mice harboring a conditional deletion at the Blm locus. BLM is RecQ helicase and is hypothesized to function in a restrictive role with respect to crossover function. In this respect, BLM and MLH1-MLH3 would act in opposition to modulate crossover frequency and distribution. The interaction between MLH1-MLH3 and MUS81 will be explored using Mus81-/- mice since this endonuclease has been shown, in yeast, to mediate an alternate crossing over pathway that is independent of MLH1-MLH3. The importance of this study is underscored by our recent observation that approximately 10% of crossovers in the mouse are MLH1-MLH3 independent, suggesting that the MUS81 pathway might functionally replace MLH1-MLH3-driven crossover mechanisms in this cohort. A second part of this aim will test interactions between EXO1, a downstream co-ordinator of MMR function, and MUS81/BLM. Finally, in aim 3, we will utilize a tandem affinity purification (TAP) system to identify protein complexes containing MLH1 and MLH3 by generating Mlh3-/- BAC transgenic mice containing a TAP-tagged Mlh3 fusion transgene. This will enable us to isolate meiotically-relevant native proteins that interact with MLH3 on meiotic chromosomes for subsequent functional analysis. Together, these studies will contribute substantially to our knowledge of meiosis and, more specifically, to the important role of MLH1-MLH3 therein. PUBLIC HEALTH RELEVANCE: Spontaneous chromosome abnormalities, which can result in serious human diseases such as Down Syndrome, are exceptionally prevalent in the human population, and are thought to arise mainly as a result of errors in maternal meiosis (the specialized cell division that gives rise to eggs and sperm). Previous studies in our laboratory have shown that a group of proteins, called the DNA mismatch repair family, are essential for normal progression of meiosis. We seek to understand more fully the role of these proteins in meiosis and to evaluate whether disruption of this pathway might explain some of the errors seen in humans. Since over a quarter of all human pregnancies are associated with such meiotic errors, this research will be essential for improving human pregnancy outcomes, most often resulting in miscarriage or birth defects, this research will have a direct impact on human health and quality of life.